The present invention relates to a process for preparing N-acetylneuraminic acid. More particularly, it relates to a process comprising isomerization of N-acetylglucosamine, in the presence of N-acylglucosamine-2-epimerase (E.C. 5.1.3.8), to give N-acetylmannosamine which is then reacted with pyruvic acid in the presence of N-acetylneuraminic acid pyruvate lyase (E.C. 4.1.3 3) to give N-acetylneuraminic acid.
N-acetylneuraminic acid (abbreviated as Neu5Ac in the following) is the most important representative of the sialic acid class of substances. Sialic acids occupy the terminal positions of glycolconjugates such as glycolipids and glycoproteins which are, for example, on the cell surfaces and carry out important functions in the differentiation, maturation and intracellular interaction of cells. The synthesis of short-chain oligosaccharides having terminal sialic acids, in particular Neu5Ac, is gaining more and more interest in this connection. The treatment of cancers by Neu5Ac derivatives has also been reported (see S. Sabesan et al., J. Am. Chem. Soc. 108: 2068-2080 (1986); R. Schauer, R. Adv. Carbohvdr. Chem. Biochem. 40: 131-234 (1982)).
Neu5Ac has hitherto been isolated from natural sources (cow's milk, swallows' nests; c.f. Schauer, ibid.). However, the availability of these sources is limited and the purification is difficult and time-consuming because of the large number of sialic acids present therein.
The enzymatic synthesis of Neu5Ac from N-acetyl-mannosamine and pyruvic acid (hereinafter abbreviated as ManNAc and Pyr, respectively) has been known since the I960s (Comb et al., J. Biol. Chem. 235: 2529-2537 (I960)). The enzyme used is N-acetylneuraminic acid pyruvate lyase (E.C. 4.1.3.3), hereinafter referred to as lyase. During this process the following reaction takes place: ##STR1##
Recent papers (M.-J. Kim et al., J. Am. Chem. Soc. 110: 6481-6486 (1988) and C. Auge et al., Tetrahedron Letters 30: 2217-2220 (1989)) report the synthesis of Neu5Ac, in which the lyase is used in immobilized form covalently coupled to insoluble carriers (PAN or agarose). With this form of immobilization, losses of activity due to the coupling are observed. A continuous production using enzyme which has been immobilized on a carrier is only possible when agents having antibacterial action are added.
The starting material for this N-acetylneuraminic acid formation is the relatively expensive N-acetylmannosamine. Availability of this starting material from N-acetylglucosamine (GlcNAc) has been mentioned in the paper by M.J. Kim referred to above. This paper considers the possibility of obtaining ManNAc by an isomerization of GlcNAc catalyzed by bases with the formation of ManNAc by a chemical method or by an isomerization of GlcNAc to ManNAc catalyzed by epimerase. No concrete data whatsoever are given, although the epimerase and its usefulness for the conversion of GlcNAc into ManNAc were known (see Ghosh et al., J. of Biol. Chem. 240: 1531-6 (1965)).